Anthrax infections are initiated by endospores of B. anthracis, a Gram-positive soil organism. Anthrax endospores do not divide, have no measurable metabolism, and are resistant to drying, heat, ultraviolet light, gamma radiation, and many disinfectants. In some types of soil, anthrax spores can remain dormant for decades. All known anthrax virulence genes are expressed by vegetative cells of B. anthracis that result from the germination of spores within the body. Endospores introduced into the body by abrasion, inhalation, or ingestion are phagocytosed by macrophages and carried to regional lymph nodes. Endospores germinate inside the macrophages and become vegetative bacteria; the vegetative bacteria are then released from the macrophages, multiply in the lymphatic system, and enter the bloodstream until there are as many as 107 to 108 organisms/ml of blood, causing massive septicemia. Once they have been released from the macrophages, there is no evidence that an immune response is initiated against vegetative bacilli. Anthrax bacilli express virulence factors, including toxin and capsule polypeptides. The resulting toxemia and bacterimia have systemic effects that lead to death of the host.
The major virulence factors of B. anthracis are encoded on two virulence plasmids, pX01 and pX02. The toxin-bearing plasmid, pX01, is 184.5 kilobases (Kb) in size and codes for the genes that produce the secreted exotoxins. The toxin gene complex is composed of protective antigen (PA), lethal factor (lef), and edema factor (EF). The three exotoxin components combine to form two binary toxins. Edema toxin consists of EF, which is a calmodulin-dependent adenylate cyclase, and PA, the binding moiety that permits entry of the toxin into the host cell. Increased cellular levels of cyclic AMP interfere with water homeostasis and are believed to be responsible for the massive edema seen in cutaneous anthrax. Edema toxin inhibits neutrophil function in vitro and neutrophil function is impaired in patients with cutaneous anthrax infection. Lethal toxin consists of LF, which is a zinc metalloprotease that inactivates mitogen-activated-protein kinase kinase in vitro, and PA, which acts as the binding domain. Lethal toxin stimulates macrophages to release tumor necrosis factor α and interleukin 1β, which are partly responsible for sudden death in systemic anthrax. The capsule-bearing plasmid, pX02, is 95.3 Kb in size and codes for three genes (capB, capC, and capA) involved in the synthesis of the polyglutamyl capsule.
The exotoxins are thought to inhibit the immune response mounted against infection, whereas the capsule inhibits phagocytosis of vegetative anthrax bacilli. The expression of all known major virulence factors is regulated by host-specific factors such as elevated temperature (>37° C.) and carbon dioxide concentration (>5%) and by the presence of serum components. Both plasmids are required for full virulence and the loss of either results in an attenuated strain. Historically, bacterial strains used for anthrax vaccines were made by rendering virulent strains free of one or both plasmids. By way of example, ‘Pasteur’ is an avirulent pX02-carrying strain that is encapsulated but does not express exotoxin components, while ‘Sterne’ is an attenuated strain that carries pX01 and can synthesize exotoxin components but does not have a capsule.
There is currently a need for reliable and rapid detection methods for anthrax bacteria. A nucleic acid based assay would be of particular interest since it could be readily adapted to high-throughput and in-field applications. Fully virulent or vaccine strains of B. anthracis can be easily identified by their virulence plasmid composition, however, many B. anthracis strains do not contain these plasmids, e.g., fully attenuated B. anthracis strains, and, as such, cannot be detected by such methods. Also, specific detection of the B. anthracis chromosome has been difficult due to its close phylogenetic relationship to other members of the B. cereus group. There have been many attempts to develop chromosomal detection assays for B. anthracis, however they are not reliable because they do not adequately distinguish B. anthracis from other Bacillus species in the B. cereus clade. In addition, B. anthracis virulence plasmids may be transferred to near neighbor B. cereus clade species, resulting in derivatives with unconventional virulence and genetic compositions which would not be distinguished by existing assays.
Accordingly, there is a need for rapid and reliable DNA-based assays for detecting anthrax and related Bacillus spp. This invention meets this need, and others.
Literature
Literature of interest includes: U.S. Patent publication 20030082563, U.S. Pat. No. 6,448,016 and Jackson et al, Proc. Natl. Acad. Sci, 95: 1224-9 (1998), Ruhfel et al, J. Bact., 157: 708-11 (1984), Patra et al., FEMS Microbiol. 15: 223-231 (1996), and Anderson et al., J. Bacteriol. 178: 377-384 (1996).